ROLE OF ATP-UBIQUITIN-DEPENDENT PROTEOLYSIS IN MHC-1 RESTRICTED ANTIGEN PRESENTATION AND INHIBITORS THEREOF

Abstract: Described herein are methods and drugs that inhibit the processing of antigens for presentation by major histocompatibility complex class I molecules. Specifically, inhibitors of the ATP-ubiquitin dependent proteolytic pathway are described, which can inhibit MHC-I antigen presentation. These methods and drugs may be useful for the treatment of autoimmune diseases and for reducing rejection of organs and graft transplants.

Description: ROLE OF ATP-UBIQUITIN-DEPENDENT PROTEOLYSIS IN MHC-1 RESTRICTED ANTIGEN PRESENTATION AND INHIBITORS THEREOF DESCRIPTION Background There are many diseases that result from the immune system attacking and destroying tissues of the body. These conditions can cause severe morbidity and in many cases can be fatal. Some of these diseases, such as systemic lupus erythematosus, result from an individual's immune system reacting against its own cells as if they were foreign or pathogenic. Collectively these diseases are referred to as autoimmune diseases. Other diseases result from the immune system responding against molecules, such as therapeutic drugs, that are present in the body's tissues. Yet other diseases are caused when the immune system responds against transplanted foreign tissues in the body. The immune response against foreign tissues iε the major cause of failure of transplanted organs or grafts in transplantation and is the principle barrier to this procedure. In these diseases, the cells that cause the destructive immune response are lymphocytes, and in the vast majority of cases, T lymphocytes play an obligatory role in these responses. The immune response iε initiated when T lymphocytes recognize fragments of antigens that are bound to major histocompatibility complex (MHC) molecules on the surface of antigen- presenting cells. The antigenic peptide bound to the MHC molecules stimulates the T cells and directs them to respond against the cells bearing the antigen-MHC complex. There are several different kinds of T lymphocytes. The two major subsets of T lymphocytes, which are distinguished by the expression of certain cell surface molecules, differ in their specificity and function. T cells that express the CD4 molecule are specific for antigens presented by MHC class II molecules and usually stimulate antibody or inflammatory responses. T cells that express CD8 molecules are specific for antigens presented by MHC class I molecules and function to kill host cells. The CD8 + T cells mediate tissue damage in several of the immune mediated diseases described above. The therapeutic approaches to treating the immune- mediated diseases are directed at blocking the response of the lymphocytes. Some therapies, such as anti- lymphocyte globulin, seek to eliminate T cells. Other therapies, such as cyclosporin A and steroids, seek to block the response of lymphocytes by inhibiting lymphocyte activation. Still other therapies, such as treatment with monoclonal antibodies against cell interaction molecules, seek to block lymphocytes from interacting with other cells. All of these therapies have significant adverse side effects. Several of the drugs have associated toxicities; e.g., cyclosporin is associated with nephrotoxicity, and steroids cause Cushing'ε disease. Monoclonal antibodies are associated with immune responses against the antibodies, which makes continued therapy ineffective. In all cases, immunosuppression results in increased susceptibility to infection, which can be lethal. A better approach to controlling or altering T cell responses would be very beneficial therapeutically. Summary of the Invention Described herein are methods and drugs for blocking cytolytic immune responses, which are useful for the treatment of a variety of autoimmune diseases and for preventing rejection of transplanted organs or grafts. These methods and drugs block the generation of cellular immunity by preventing the initial presentation of cellular and viral antigens on MHC-class I molecules to T cells. The methods comprise using inhibitors of the ATP-ubiquitin-dependent proteolytic pathway to prevent or reduce the processing of intracellular proteins into the antigenic peptides that bind to MHC-1 molecules. This invention results from Applicants' work, which shows that inhibition of an early step (ubiquitin activation) and a late step (cleavage of intracellular proteins into peptides) in the ATP-ubiquitin-dependent proteolytic pathway can inhibit MHC-1 antigen presentation by blocking the generation of peptides binding to MHC-1 molecules. This work provides strong evidence for the role of this cytosolic protein degradation system in the processing of MHC-1 presented antigens for MHC-1 presentation. Furthermore, Applicants have discovered new features of the function of the proteoso e (multicatalytic protease complex) , an essential component of the ATP-ubiquitin pathway. As a result of their work, methods and drugs are provided for reducing cytolytic immune responses without affecting antibody-mediated or inflammatory responses. Thiε invention should thus provide therapeutics useful for autoimmune diseases and prevention of transplant rejection without the attendant disadvantages of generalized immune suppression. Specifically, Applicants have discovered that gamma interferon (γ-IFN) , which has long been known to stimulate antigen presentation, alters the relative activities of the multiple peptidases of the proteasome, so as to promote the generation of peptides with the characteristic carboxyl-terminal residues found on MHC-1 bound peptides. The trypsin-like peptidase, which cleaves on the carboxyl side of basic residues, and the chymotrypsin-like peptidase, which cleaves after hydrophobic residues, were found to have increased activity, while the peptidylgluta yl peptidase, which cleaves after acidic residues, had decreased activity. Furthermore this effect of γ-IFN on the proteasomes iε dependent on the expression of two genes at the MHC locus, LMP 2 and LMP 7. Secondly, Applicants have demonstrated that MHC-I antigen presentation can be blocked by inactivation of ubiquitin conjugation, an early step in the ATP- ubiquitin-dependent proteolytic pathway, It was found that, at the nonper issive temperature, clasε I- restricted antigen presentation was inhibited in a mutant cell line expressing a thermolabile ubiquitin activating enzyme (El) . Applicants have further demonstrated that chymostatin, an inhibitor of chymotrypsin-like proteases, inhibits MHC-I presentation by antigen presenting cells. Other inhibitorε, particularly of the chymotrypsin-like and trypsin-like activities of proteasomes, are further described herein. Brief Description of Drawings Figure 1 shows the effect of gamma interferon (γ- IFN) on peptidase activities of proteasomes from U937 cellε. The upper panel shows the hydrolysis of the indicated substrates by proteasome fractions from control cells and cells treated with γ-IFN. The lower panel shows kinetic analysis of rates of cleavage of the subεtrates by the same proteasome fractions. Figure 2 εhowε the effect of γ-IFN on peptidase activities of purified 20S and 26S proteasomes. Figure 3 shows the difference in activities of the peptidaεeε of proteaεomes from wild-type and MHC- deficient lymphoblastoid cells. The upper panel εhowε the peptidaεe activities. The lower panel shows kinetic analysiε. Figure 4 εhowε the frequency of amino acid reεidues preceding bonds cleaved to generate peptides presented on MHC-claεε I moleculeε. The left panel shows the frequency of amino acids at the carboxyl-termini. The right panel showε the frequency of amino acidε preceding the amino-terminal reεidueε. Figure 5 (A-D) shows MHC class I-restricted preεentation of ovalbumin (OVA) by mutant (tε20.10.2) and wild-type (E36.12.4) cellε. Figure 6 (A-B) εhowε the effect of temperature on the synthesiε and maturation of H-2K b MHC heavy chainε. Figure 7 (A-B) shows MHC clasε I-reεtricted preεentation of endogenously synthesized OVA 257 _ 264 peptide. Figure 8 shows inhibition of MHC clasε I-reεtricted preεentation of ovalbumin (left) and ovalbumin peptide (right) by chy oεtatin. Detailed Description This invention relateε to an approach for inhibiting cytolytic immune responses that avoids generalized εuppreεεion of T lymphocyte function with itε attendant risks of infection. The methods and drugs of this approach are useful for treating autoimmune diseases and preventing rejection of foreign tissues, such as transplanted organs or grafts. The strategy is to inhibit antigen presentation by major histocompatibility complex (MHC) I molecules rather than suppress T cell activity. This approach haε the advantage that it selectively affects only clasε I MHC- restricted immune reεponεeε and not antibody or other CD4 + T cell-mediated responses. Consequently, there should be less generalized immunosuppresεion and susceptibility to infection in the patient. Drugs that interfere with clasε I antigen presentation should also be useful in combination with other existing therapies and may lower the dose used in these therapies, which would decrease toxicities. Specifically, methods and drugs are described that inhibit the processing of internalized cellular or viral antigens into the kind of peptides, referred to as antigenic peptideε, that bind to the MHC-I moleculeε. MHC-I binding peptides have strict sequence and size requirements. In the absence of the antigenic peptideε, the antigens are not presented at the cell surface and CD8 + T cells are not stimulated. This method εhould block both the initiation of immune reεponse and stop ongoing immune responseε. Thiε invention is based on Applicants' work (see Examples below) supporting the role of the ATP-ubiquitin dependent proteolytic pathway, and in particular, proteaεomes (multicatalytic proteinase complexeε) , in the generation of MHC-I-aεsociated antigenic peptides. An initial step in the presentation of intracellular and viral proteins to the immune system iε their proteolytic degradation in the cytosol to small peptides (Townεend and Bodmer, 1989) . The antigenic fragments are then taken up via specific membrane transporters (Monaco, 1992; Powis, et al. , 1991; Spies and DeMars, 1991) into the endoplasmic reticulum, where they associate with the MHC-I moleculeε. These peptide-protein complexes are then transported to the cell surface for presentation to cytotoxic T cells. The cleavage of proteins into antigenic peptideε εeemε to be a regulated proceεs, since there are strict size and sequence constraints on the peptides that can bind to MHC-class I moleculeε. The identity of the cleavage enzymes and the nature and precise location(ε) of the proteolytic steps that generate the 8-9 residue peptides presented on MHC-claεε I molecules have not been definitively eεtabliεhed, although it iε clear that the proteolysis does not occur in lysosomal or endosomal compartments (Morrison et al . . 1986) . Recent εtudieε have implicated the proteasomes, an esεential component of the ATP-ubiquitin-dependent proteolytic pathway, in the generation of antigenic peptideε. Several proteolytic εystems exist in eukaryotic cells, and the ATP-ubiquitin pathway is the major cytosolic pathway of protein degradation (Finley and Chau, 1991; Hershko and Ciechanover, 1982; Rechεteiner, 1987) . In this multistep proceεε, protein substrates are first modified by covalent conjugation to multiple ubiquitin moieties, which marks them for rapid degradation by a 26S (1,500 kD) ATP-dependent proteolytic complex, called the 26S proteoεo e or UCDEN (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992; axman et al . , 1987; Hough et al . , 1987) . This large structure contains the 20S (about 700 kD) proteasome as its proteolytic core pluε many additional regulatory and catalytic co ponentε (Goldberg and Rock, 1992; Orlowεki, 1990; Rivett, 1989; Waxman et al ., 1987; Hough et al ., 1987; Armon et al . , 1990; Driscoll and Goldberg, 1990; Eytan et al . , 1989). The precise intracellular function of the 20S proteosome is not clear; when iεolated, this particle can degrade proteins and oligopeptides, but not ubiquitin-conjugated proteins (Goldberg, 1992; Tanaka et al . , 1992; Armon, 1990; Driscoll and Goldberg, 1990a; Eytan et al . , 1989; Driεcoll and Goldberg, 1989) . The 20S proteaεome is composed of about 15 distinct subunits of 20 - 30 kD. It contains three or four different neutral peptidases, which cleave specifically on the carboxyl side of hydrophobic, basic and acidic amino acidε (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992; Orlowski, 1990; Rivett, 1989) . These peptidases are referred to as the chymotrypsin- like peptidaεe, the trypsin-like peptidase, and the peptidylglutamyl peptidase, respectively. Which subunits are reεponεible for theεe activitieε iε unknown, although the cDNAs encoding several subunits have been cloned (Tanaka et al . , 1992). Recent studieε have found that the 20S proteasomes resemble in εize and subunit composition the MHC-linked low molecular weight protein (LMP) particles (Driscoll and Finley, 1992; Goldberg and Rock, 1992; Monaco and McDevitt, 1986; Parham, 1990; Martinez and Monaco, 1991; Oritz-Navarette et al . , 1991; Glynne et al . , 1991; Kelly et al . r 1991; Monaco and McDevitt, 1982; Brown et al . , 1991; Goldberg, 1992; Tanaka et al . , 1992) . The LMP particleε contain two polypeptideε, LMP 2 and LMP 7, which are encoded in the MHC chromosomal region. Treatment of cellε with gamma interferon (γ-IFN) stimulates antigen presentation (Townsend and Bodmer, 1989; Yewdell and Bennink, 1992) and causes the induction of LMP 2 and LMP 7 , as well aε other MHC genes (Glynne et al . , 1991; Kelly et al . , 1991; Monaco and McDevitt, 1982; Yang et al . , 1992). Immunochemical studies strongly suggest that LMP 2 and LMP 7 are two subunits of particles representing a small fraction of the 2OS proteosome population. However, the importance of these subunitε in the immune reεponse is uncertain, εince deletion of theεe geneε does not prevent antigen presentation, in contrast to deletion of other MHC-geneε (Arnold et al . , 1992; Mamburg et al . , 1992) . Moreover, γ-IFN can alter the polypeptide composition of 20S and 26S proteasomeε (Yang et al. , 1992) in mutantε lacking the LMP 2 and 7. It haε, thuε, been a matter of uncertainty whether proteaεomeε play a role in antigen preεentation (see diεcusεionε in Goldberg and Rock, 1992; Driscoll and Finley, 1992; Yewdell and Bennink, 1992, pp. 26-28) . Applicants' work described herein provides confirming evidence for a role for the 20S and 26S proteasomeε and the ATP-ubiquitin proteolytic pathway in the generation of antigenic peptides for MHC-1 antigen preεentation. In addition, thiε work reveals new features of the pathway of proteolysis of antigens and of proteasome function, which led to the present invention. Firstly, they have shown that gamma interferon, which is known to εtimulate antigen presentation, εelectively enhances those peptidase activities, namely, the trypsin-like and chymotrypsin- like activities, of the proteasomeε that produce peptideε that can bind to MHC-1 molecules, i.e. peptideε with baεic and hydrophobic carboxyl-terminal reεidueε (Example 1) . Gamma interferon increased 2- to 6-fold the capacity of purified 2OS and 26S proteasomes to cleave peptides after hydrophobic and basic reεidueε, while reducing cleavage after acidic reεidues. They have further shown that this enhancement by gamma interferon is dependent on the expreεεion of the MHC gene productε, LMP 2 and LMP 7. Their findings, thus, provide evidence to support the role of proteasomes in antigen presentation. Furthermore, they indicate that the three peptidases of proteasomes are distinctly regulated, that changeε in the relative activities of the peptidases can alter the nature of peptides that are generated by the proteasome and which are available for MHC-1 antigen presentation, and that the MHC-encoded subunits are involved in the regulation of the peptidase activities. Secondly, Applicants have shown that a defect in ubiquitin conjugation, an early step in the ATP- ubiquitin-dependent proteolytic pathway, leads to reduced MHC-I-reεtricted antigen presentation (Example 2) . Using cells exhibiting a temperature-senεitive defect in the ubiquitin activation enzyme. El, they found that the nonpermissive temperature inhibited class I-restricted presentation of ovalbumin (OVA) introduced into the cytosol, but did not affect presentation of an OVA peptide syntheεized from a minigene. Theεe findingε provide evidence for the role of the ATP-ubiquitin proteolytic εyεtem in MHC-I antigen preεentation. They de onεtrate that a block at either an early εtep (ubiquitin conjugation) or a late εtep (proceεεing of carboxyl-termini of the peptideε) in the proteolyεiε of intracellular proteinε by the ATP- ubiquitin system can inhibit clasε I restricted presentation. It is reasonable to expect that inhibition of other steps in this proteolytic pathway will alεo produce a εi ilar effect. Applicants' diεcoverieε of these features of proteosome function and their demonstration that inhibition at a single step in the ATP-ubiquitin pathway can inhibit antigen presentation lead to the present invention. It is reaεonable to expect that inhibitors that block single or multiple steps of thiε proteolytic pathway would inhibit MHC-I-restricted antigen presentation, and that such inhibitors would be useful in reducing CD8 + mediated cytolytic immune responses. Such inhibitors should be useful for therapy or prevention of autoimmune diseases and for reducing rejection of transplanted and other foreign tissues. Inhibitors of either or both the trypsin-like and chymotrypsin-like peptidases of proteasomeε and inhibitorε of ubiquitin conjugation are particularly expected to be useful for these purposes. As described herein, Applicants have demonεtrated the feaεibility of thiε invention by εhowing that chymoεtatin, which selectively inhibits cleavage after hydrophobic residueε, inhibitε the MHC-I preεentation of ovalbumin (Example 3). Antigen preεentation of an OVA peptide introduced into the cytosol was not inhibited, indicating that chymoεtatin iε affecting the processing of the OVA protein into the peptide. This and other inhibitorε are further described in the following section. Inhibitorε of the ATP-Ubiσuitin Dependent Proteolytic Pathway Various inhibitors of the peptidases of proteasomeε have been reported (see, e.g., Tanaka et al . , 1992; Orlowski, 1990; Goldberg, 1992; Rivett et al . , 1989a [Arch. Biochem. Biophyε. 268:1-8] and 1989b [J. Biol. Chem. 264:12,215-12219]; Dick et al . , 1991). These include known inhibitors of chymotrypsin-like and trypsin-like proteases, as well as inhibitorε of thiol (or cysteine) and serine proteaseε. In addition, some endogenous inhibitorε of proteosome activities have been isolated. These include the 240 kD and the 200 kD inhibitorε iεolated from human erythrocytes (Murakami and Etlinger, 1986; Li et al . , 1991 and purified CF-2 (described in Example 4; Goldberg, 1992). As exemplified by these known inhibitorε of proteasomes, other molecules, including known inhibitors of chymotrypsin-like, trypsin-like, thiol, and serine proteases, can be tested using assays for the proteosome peptidases (see Exampleε) . The inhibitorε can be naturally iεolated or synthetic and peptide or non-peptide molecules. Preferably, the inhibitors would selectively inhibit the chymotrypsin-like and trypεin-like peptidaεeε, while leaving the peptidylglutamyl peptidaεe relatively unaffected. A typical inhibitor would be a peptide aldehyde, like leupeptin, which inhibitε primarily cleavage after baεic residues, or chymostatin, which inhibitε primarily after hydrophobic residues. In addition to these antibiotic inhibitors originally isolated from Actinomyceteε (Aoyagi and Umezawa, 1975) , a variety of peptide aldehydeε have been εyntheεized, such as the inhibitors of chymotrypsin-like proteases described by Siman et al . (WO 91/13904) . Novel moleculeε can also be obtained and tested for inhibitory activity. Aε illuεtrated by the above cited referenceε, various strategies are known in the art for obtaining the inhibitors for a given protease. Compound or extract libraries can be screened for inhibitors using peptidase assays. Alternatively, peptide and peptidomimetic molecules can be designed based on knowledge of the substrates of the protease. For example, substrate analogs can be synthesized containing a reactive group likely to interact with the catalytic site of the protease (see, e.g., Siman et al . , WO 91/13904; Powers et al . , 1986). The inhibitors can be stable analogs of tranεition intermediateε (tranεition state analogs), such as Cbz-Gly-Gly-leucinal, which inhibits the chymotrypsin-like activity of the proteosome (Orlowski, 1990; see also Kennedy and Schultz, 1979) . Variantε or analogs of known inhibitorε, such as chymostatin, can be also be εynthesized. Various natural and chemical proteaεe inhibitorε reported in the literature, or moleculeε εimilar to them, are likely to inhibit the activity of the two proteaεomal peptidases. These include peptides containing an α-dicarbonyl unit, such aε an α-diketone or an α-keto ester, peptide chloromethyl ketones, isocou arins, peptide sulfonyl fluorides, peptidyl boronates, peptide epoxides and peptidyl diazomethanes (Angelaεtro et al . , 1990; Bey et al . , EPO 363,284; Bey et al . , EPO 364,344; Grubb et al . , WO 88/10266; Higuchi et al . , EPO 393,457; Ewoldt et al . , 1992; Hernandez et al . , 1992; Vlaεak et al . , 1989; Hudig et al . , 1991; Odakc et al . , 1881; Vijayalakshmi et al . , 1991; Kam et al . , 1990; Powers et aJ., 1989; Oweida et al., 1990; Powerε et al . , 1990; Hudig et al . , 1989; Orlowski et al . , 1989; Zunino et al., 1988; Kam et al., 1988; Parkes et al . , 1985; Green and Shaw, 1981; Angliker et al . , 1987; Puri et al . , 1989; Hanada et al . , 1983; Kajiwara et al . , 1987; Rao et al . , 1987; Tsujinaka et al . , 1988]. Various inhibitors of ubiquitin conjugation to proteins are also known (see Wilkinεon et al . , 1990) . Ubiquitin conjugation itself iε a multistep process, involving the activities of an ubiquitin activating enzyme (El) , a group of ubiquitin-carrier proteinε (E2) , which catalyze the tranεfer of ubiquitin to target proteinε, and the ubiquitin-protein ligaεe, E3. Moleculeε that block ubiquitin conjugation at any of these εtepε are alεo expected to be inhibitorε of antigen preεentation. Preferably, εpecific inhibitors of these enzymes will be used, such as εubεtrate and tranεition state analogs. For example, adenoεyl- phoεpho-ubiquitinol, an analog of the subεtrate ubiquitin adenylate, has been found to be a εpecific and effective inhibitor of El (Wilkinson et al . , 1990). The inhibitors can be uεed in vitro or in vivo to block MHC-I antigen preεentation. They can be adminiεtered by any number of known routes, including orally, intravenously, intramuεcularly, topically, and by infuεion (see, e.g., Platt and Stracher, U.S. Patent 4,510,130; Badalamente et al . , 1989; Staubli et al . , 1988). Preferably, the inhibitors are low molecular weight molecules. Suitable vehicles for protein drug delivery, such as liposomes, may alεo be used. Vehicles than can target the drug to εpecific tissues can also be uεed. Examples The following Examples further describe Applicants' work in more detail and more specifically illustrate this invention. Tableε are located at the end of each Example. Example 1 Regulation of the Peptidase Activities of Proteasomes Bv γ-IFN and MHC Genes The present εtudieε were undertaken to clarify the role of γ-IFN, proteasomeε, and MHC-encoded proteinε in the proceεεing of cell antigenε. The work deεcribed below demonεtrateε that the pattern of peptidaεe activitieε of the 20S and 26S proteasome complexes iε altered following treatment of cells with γ-IFN and upon expression of the MHC region that contains the LMP 2 and 7 genes. These changes in catalytic activities should favor the generation of the types of peptide sequences (Falk et al . , 1991) that bind specifically to MHC-claεε I proteins. γ-Interferon Alters Proteosome Activities. To learn whether γ-IFN regulates the function of proteaεomes, several hydrolytic activities of the proteasomeε and of εoluble extracts from control U937 cells (a human monocytic line) and U937 cells treated with γ-IFN were examined. Although this treatment caused a large induction of MHC-clasε I genes and proteins encoded in the MHC region (i.e. LMP 2 or TAP 1) , no significant difference waε found in the ability of iεolated proteaεo es or cell extracts to degrade 125 I-lactalbumin or ubiquitinated 125 I-lactalbumin to acid-soluble fragments. However, clear changes were found after γ-IFN treatment in the ability of the proteasomes to hydrolyze different peptide substrates. Fluorogenic peptide substrates were used to measure the hydrolytic activities of proteasomeε: SUC-LLVY-MCΛ [SEQ ID No: 3] (hydrophobic) for the "chymotrypεin-like" peptidaεe, Boc-LRR-MCA (baεic) for the '-trypsin-like" peptidaεe, and Cbz-LLE-MNA (acidic) for the peptidylglutamyl peptidase. Proteaso e-enriched fractions, as well as crude extracts, from γ-IFN-treated U937 cells hydrolyzed the hydrophobic peptide, Suc-LLVY-MCA, 2- to 3-fold faster and the baεic peptide, Boc-LRR-MCA, 3- to 4-fold faster than εimilar preparations from control cells (Figure 1) . These effects of γ-IFN-treatment were seen in the absence of ATP. Kinetic analysis indicated that γ-IFN caused a 2-fold increase in the maximal capacity (V maχ ) of the proteasome to hydrolyze the hydrophobic substrate and a 6-fold increase in V [naχ with the basic substrate (Figure 1) . In addition, a small increase in the K--.S for these substrates (averaging 50%) was found consistently, but such an effect cannot account for the accelerated peptide hydrolysis (Figure 1) . Very similar changes in V max were seen in analogous studies using purified 20S proteasome (see below) . The cleavage of a variety of other hydrophobic and baεic peptides, which are degraded more slowly than Suc-LLVY-MCA or Boc-LRR- MCA (respectively) , increased in a similar fashion after γ-IFN treatment, as did hydrolysis of these standard substrates (Table 1) . Thus, proteasomes from γ-IFN- treated cells are generally more efficient in cleaving after hydrophobic and basic residues, although the εpecificitieε of theεe active sites do not appear to change. Under these conditions, the overall capacity of the cellε to hydrolyze these basic and hydrophobic peptides increases. However, no change was found in total proteasome content. The ability of the crude extracts to hydrolyze these peptides changed in a similar fashion, as did proteasome fractions, which contain about 80% of the activities againεt theεe substrates in post-nuclear cell extracts. Moεt of the remaining peptidase activities (15% total) was due to proteaεomeε that were tightly associated with microsomal membranes, and γ-IFN treatment also enhanced the cleavage of Suc- LLVY-MCA and Boc-LRR-MCA by proteasomeε in the microsomal and nuclear fractions. It is noteworthy that not all of the proteasomeε'ε peptidaεe activitieε increase after γ-IFN treatment. Proteaεomal fractions and the crude extracts from γ-IFN- treated U937 cells cleaved the acidic εubεtrate, Cbz- LLE-MNA (at 100 mM) , at roughly similar rates aε controls. Further kinetic analysis, however, indicated that there was a reproducible decreaεe of approximately 30% in the V max value for thiε substrate (Figure 1) (as well as a decrease in the K,,,) . Thus, the εeveral catalytic εites of the proteasome are regulated in distinct fashionε by γ-IFN. Behavior of 2OS and 26S Complexes Within cellε, proteaεomes are found either as part of the 26S (1,500 kD) complexes, which degrade preferentially ubiguitin-conjugated proteins, or as 20S particles, which by themselves cannot hydrolyze Ub- conjugated proteins, despite having multiple proteolytic activitieε (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992; Armon et al . , 1990; Driεcoll and Goldberg, 1992; Eytan et al . , 1989; Driεcoll and Goldberg, 1989; Matthews et al . , 1989) . To examine whether γ-IFN alters the catalytic activitieε of both the 2OS and 26S proteasomes, these εtructureε were purified by FPLC uεing anion exchange and gel filtration columns (Figure 2) . A good separation of the 20S and 26S forms of the proteasome was achieved. Native PAGE of the 20S fraction showed the characteristic 700 kD band, while the 2OS sampleε contained a 1500 kD band pluε some 700 kD material. Aε expected, theεe 26S preparations degraded ubiquitinated- 125 I-lactalbumin in the presence of ATP, but exhibited little or no activity against free 125 I-lactalbumin. In contrast, the 20S fraction degraded this protein, but not if it was ubiquitinated. Both 2OS and 26S forms exhibited the three peptidase activities, which could be stimulated several-fold by ATP. ATP (2 mM) εtimulated the degradation of Suc-LLVY-MCA by 2OS proteaεomes and the degradation of the same substrate 5-fold by 26S proteasomeε from control cellε. In the preεence of ATP, εignificant differenceε between control and γ-IFN- treated preparationε were retained for 2OS proteaεomes.. Interferon-treatment caused similar changes in the peptidaεe activities of both 20S and 26S proteasomeε, aε were found with cruder preparationε. With both formε, cleavages of hydrophobic and baεic εubεtrateε increased significantly with γ-IFN, although the percent increase tended to be somewhat smaller for the 26S fraction. Interestingly, cleavage of the acid εubεtrate by either particle did not increase, and its hydrolysiε by the 26S particle decreaεed by 50% after γ-IFN treatment (Figure 2) . By contrast, after γ-IFN treatment, no conεiεtent differenceε were obεerved in the abilities of the 20S or 26S particles to degrade free 125 I-lactalbumin or ubiquitinated 125 I-lactalbumin either in the absence or presence of ATP. Taken together, these findings suggest that γ-IFN alters the pattern of cleavages in polypeptides made by both 2OS and 26S proteasomes, without affecting the overall rates of protein digestion. Proteasome Activities in MHC-Deficient Cells Since two proteins encoded in the MHC region, LMP 2 and LMP 7, associate with proteasomeε (Driεcoll and Finley, 1992; Goldberg and Rock, 1992; Monaco and McDevitt, 1986; Parham, 1990), and εince γ-IFN stimulates the expreεsion of theεe componentε, Applicants investigated whether deletion of this part of the MHC-locuε might influence the peptidaεe activitieε of proteasomes and their response of γ-IFN. For this purpoεe, they isolated the proteasomal fraction from 721 human B lymphoblaεtoid cells and from 721.174, a variant of these cells, that carries a homozygous deletion in the MHC region that includes the LMP 2 and LMP 7 genes (DeMars et al . , 1985) . Proteasomes from the mutant cells were found to degrade the basic subεtrate at approximately 40% of the rate and the hydrophobic subεtrate at 70% of the rate of the wild-type cellε (Figure 3) . maχ valueε for the two substrates decreased by more than 50% for proteasomeε from mutant cellε (Figure 3) . The total content of the proteasomes appeared similar in the two cell types, and no clear difference was seen in the ability of the proteaεomeε to degrade 125 I-lysozyme or ubiquitinated- 125 I-lysozyme to acid-εoluble productε. Furthermore, the breakdown of several more slowly degraded hydrophobic and basic substrates (the same ones studied in Table 1) was also lower in the MHC-deleted mutant by 40% to 80. In contrast to the reduction in these peptidase activities, proteasomeε from the MHC-deleted variant conεistently cleaved the acidic substrate, Cbz-LLE-MNA, about 40% faster than similar preparations from wild-type cellε (Figure 3) . A small, but consistent, increase in the V maχ waε seen for this subεtrate (Figure 3) . Similar changeε in the three peptidaεe activities were found in crude extracts from the mutant and wild- type cells and proteasome microsomal or "nuclear" fractions. Thus, the peptidaεe capacity of proteasomes in all fractions of the cell appear to be regulated by MHC-encoded genes and by γ-IFN. The findingε that expreεεion of MHC-encoded geneε increaεes basic and hydrophobic cleavages by the proteasome, while suppresεing acidic cleavages, parallels the resultε obtained when U937 cells were treated by γ-IFN. Theεe observations raise the possibility that the γ-IFN- dependent changes in proteasome activities are mediated, at least in part, through the expresεion of MHC-encoded proteinε, moεt likely the LMP 2 and LMP 7 proteasome subunits. To examine this hypothesis. Applicants used the 721 and 721.174 variant to investigate whether γ-IFN treatment could alter these peptidaεe activitieε in the absence of the LMP genes. In wild-type cells treated with γ-IFN, there was an increased rate of degradation of hydrophobic and basic substrates and a reduced capacity to cleave the acidic substrate. Thuε, the proteasomes from wild-type lymphocytes responded in a manner similar to those of U937 cellε (although the relative changes appeared larger in the monocyte line) . However, γ-IFN treatment of the MHC-deleted cells did not alter at all the degradation of the basic substrate, Boc-LRR-MCA, in contrast to the large changes εeen with proteaεomes from wild-type cells. In the 721.174 mutant, γ-IFN caused a much smaller increase in cleavage of the hydrophobic peptide, Suc-LLVY-MCA, than in the wild-type parent cells. These differenceε between the cells were evident with a number of different basic and hydrophobic peptides (Table 2) . Surprisingly, in mutant lymphoblastε, γ-IFN actually enhanced hydrolyεiε by proteasomes of the acidic substrate (Table 2) , even though in the wild-type parent cells (and in the U937 line) , γ-IFN reduced reproducibly cleavage of this substrate. Clearly, the IFN-induced alterations in the activities of the three catalytic sites of the proteasome, either directly or indirectly, are MHC- dependent. Protein Breakdown and Proteasome Function Two general new concluεionε have emerged from these experiments: 1) that the catalytic functionε of proteaεomeε are qualitatively regulated by gamma- interferon and theεe actionε require the preεence of MHC-encoded geneε; and 2) that the different peptidaεe εites of these particles can be regulated in distinct fashionε. After γ-IFN treatment, proteaεomes in monocytes and lymphoblasts, and presumably in many other cells, show a greater capacity to hydrolyze peptides following hydrophobic and baεic reεidueε, but a reduced capacity to cleave after acidic residues. Previouε studies have demonstrated several cellular factors that can bind reversibly to the proteasome and stimulate (Dubiel et al ., 1992; Ma et al . , 1992b) or inhibit (Driscoll et al . , 1992; Li et al ., 1991) its three peptidase activitieε coordinately (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992) . In contraεt, γ-IFN affects each of these catalytic siteε differently, presumably because it regulates gene expreεεion and thereby alters the subunit composition of the proteasome (see below) . Interestingly, γ-IFN waε not found to alter the rateε of digeεtion of a protein subεtrate, 125 I-lactalbumin or ubiquitin-conjugated 125 ι- lactalbumin to acid-soluble fragmentε by either the 2OS or 26S proteasome. These findings suggest that in vivo , the initial, presumably rate-limiting, cleavages of polypeptides or ubiquitinated proteins by proteasomes are not altered by γ-IFN. Instead, this cytokine appears to affect the later steps in this pathway, i.e. the subsequent cleavages of the acid-soluble protein fragments to short peptides. Thuε, the changeε in the relative activitieε of the three peptidases should alter the nature of the oligopeptides that are generated by the proteaεome and are available for further proteolytic proceεεing and/or delivery to MHC-class I molecules (see below) . It will be important to define the structural changes in the protease that lead to the increased rateε of hydrolysis after basic and hydrophobic sequenceε and to the decreaεed cleavage after acidic residues. The major alterations in these activities induced by γ-IFN clearly require the presence of the portion of the MHC region containing the LMP 2 and 7 geneε. Deletion of thiε region by itεelf (without γ-IFN treatment) cauεed the reverse changeε in peptidaεe activitieε of those induced by γ-IFN. Furthermore, γ-IFN is known to stimulate the expresεion of the two MHC-encoded subunits, LMP 2 and LMP 7, and to increase the abundance of "LMP particles" in cells (i.e. those proteasomes containing LMP subunits) . The simplest explanation for these findingε is that the γ-IFN-induced alterations in catalytic properties result from incorporation into proteasomes of these MHC-encoded subunits. The LMP 2 and/or LMP 7 proteins may themselves contain "tryptic- like" or "chymotryptic-like" sites. If so, these sites may supplement the peptidase sites present in the absence of γ-IFN or may replace them with sites of a higher catalytic efficiency. In this content, it iε intriguing that, in yeast, the "chymotryptic-like" activity of proteasomes is inactivated by point mutations in a proteasome-subunit gene very homologous to LMP 2 (Driεcoll et al ., 1992; Li et al . , 1991; Ma et al ., 1992) . Alternatively, incorporation into proteaεomeε of LMP 2 or LMP 7 might enhance the activity of other subunits that contain the trypsin-like or chymotryεin-like catalytic sites. Similarly, incorporation of these MHC-encoded subunits may replace or reduce the activity of subunits that comprise the peptidylglutamyl catalytic site of the proteasomes. Although these findings suggest functions for the LMP-encoded εubunits, γ-IFN clearly induce some changeε in proteaεome activity in the mutantε lacking LMP 2 and LMP 7 geneε. In these cells, γ-IFN caused a small (but reproducible) increase in the chymotryptic activity. In addition, the peptidylglutamyl activity in the mutants increases upon γ-IFN treatment, even though in wild-type cells, this activity decreases. These functional changes must ariεe through other alterationε in proteaεome subunit composition, not involving LMP 2 and LMP 7. Presumably, γ-IFN can reduce or suppress the synthesis of subunits encoded outside of this portion of the MHC region or may otherwise modify proteasome structure. In fact, Peterson and coworkers (Yang et al . , 1992) have reported that γ-IFN alterε the pattern of proteaεome subunits in mutants lacking the LMP geneε. It iε noteworthy that LMP 2 and LMP 7 proteinε are preεent in only a subset of the cell's proteasomes (Brown et al . , 1991), and within this subset, some particles may exist that contain only one of these subunits, while others may contain both. In either case, the LMP 2- and LMP 7-containing populations probably differ to a very large degree from the preexistent population in their hydrolytic activities, since the induction of theεe subεetε or proteaεomes by γ-IFN were observed to cause large changes in the total peptidaεe activitieε in the cell within 3 dayε. Thus, the changes in catalytic activity found here probably undereεtimate the actual differenceε between the activitieε of LMP-containing and LMP-deficient εubεetε of proteaεomeε. In the paεt, the proteasome has generally viewed as a single type of structure with characteristic enzymatic propertieε. This view is clearly simplistic or incorrect in light of the present findings, which emphasize the functional heterogeneity and plasticity of these particles. These findings suggeεt that in many cellε, proteaεome propertieε change in vivo during infectionε or εepεiε or other conditions, in which γ-IFN levelε riεe and immune reεponses are activated. Theεe alterations in proteasome propertieε must depend alεo on dosage and duration of exposure to thiε cytokine. The existence of functionally distinct subclasses of proteasomeε in cells may also be important in many other biological contexts. In fact, there have been reportε of alterationε in proteasome-subunit composition during cell development (Ahn et al . , 1991; Scherrer, 1990), which presumably result in distinct changeε in function and have different physiological consequences from those reported here. Implications for Antigen Presentation As part of normal protein turnover, most of the peptides generated by the proteasomeε in cytosol or nucleus must be rapidly digested to free amino acids by non-proteasomal peptidases. However, a fraction appears to be transported into the endoplasmit reticulum (ER) by the TAP 1-2 complex and to bind to MHC-class I molecules, either directly or after further proteolytic processing (Townsend and Bod er, 1989; Yewdell and Bennink, 1992; Monaco, 1992; Powis et al . , 1991; Spies and DeMarε, 1991) . The changes in the proteaso al activitieε induced by γ-IFN or reεulting from the MHC- deletion should alter the peptides generated by the proteasome during degradation of cellular and viral proteins, and thus change the repertoire of peptides available for presentation to T cells. These alterations in the relative rates of cleavage after baεic, hydrophobic, and acidic reεidueε εhould reεult in production of a different group of peptideε or of the same peptides, but in very different proportions. Specifically, the 2- to 6-fold increase in the "chymotryptic-like" and "tryptic-like" activities and the decrease in peptidylglutamyl activity should generate more short peptides ending with basic or hydrophobic carboxyl termini, and fewer peptides ending with acidic carboxyl termini. These changeε in catalytic propertieε with γ-IFN or expression of MHC-encoded geneε would thus appear to favor the generation of just those types of peptides that are known to bind to MHC-class I molecules. The C- terminal reεidue of antigenic peptideε plays a key role in their binding to specific MHC-clasε I molecules (Falk et al . , 1991; Guo et al . , 1992; Hunt et al . , 1992; Jardetzky et al . , 1991; Matsumura et al . , 1992; Parham, 1992) , as iε alεo evident from the X-ray analysis of several MHC-protein-peptide complexes (Rotzschke and Falk, 1991; Fremont et al . , 1992; Silver et al . , 1992; Zhang et al . , 1992) . It is indeed striking that the vast majority of naturally-processed peptides that have been found associated with class I molecules have hydrophobic or basic C-termini, and virtually none haε an acidic C-terminuε (Figure 4) . Thiε conclusion is based upon Applicants' survey of the published literature of MHC-associated peptides. Furthermore, an even stronger preference for hydrophobic C-termini is evident in the sequences of the average population of peptides that can be eluted from two εpecific subtypes of MHC-clasε I proteinε. Thiε predominance of baεic and hydrophobic residues was not seen at the amino acids preceding the N-termini of MHC-aεεociated peptideε (Figure 4) . Several immunological roles have been poεtulated for LMP 2 and LMP 7 εubunitε. It haε been propoεed that the LMP εubunitε direct the proteaεome particles to the endoplaεmic reticulum (ER) , where they would be in contiguity with the TAP-1/2 transporters. However, Applicants and others (Yang et al . , 1992) have found no evidence that the loss of LMP genes or γ-IFN-stimulation of LMP expresεion affectε the εubcellular diεtribution of proteaεomeε. Instead, Applicants have found that the γ-IFN-treatment and MHC-deletion change the peptidaεe activities of proteasomes in the soluble fraction of the cell, as well as the activities of the minor population of proteasomes associated with ER (10% total) and with the nuclear fraction. For transport into the ER, these antigenic peptides generated by soluble proteasomes must somehow withstand complete hydrolysis by cytosolic exopeptidases. More recently, it has been argued that LMPs do not play an important role in antigen presentation (Arnold et al . , 1992; Mamburg et al . , 1992) , because MHC-deleted cells (like 721.174) can present antigens if they are tranεfected only with the genes for the TAP-1 and -2 transporters. This work εhowε, however, that the proteasomes isolated from these mutant cells still exhibit the tryptic- and chymotryptic-like activities. These findings may explain why the MHC-deleted cellε can εtill preεent antigens on clasε I moleculeε. The reεults lead to the prediction of a decrease in the efficiency of antigen processing and presentation by the MHC-deleted mutant cells. This was in fact reported by Hammerling and coworkers (Mamburg et al . , 1992). On the other hand, the actions of γ-IFN to increase expreεεion of LMPs and thereby to change the pattern of peptides produced by the proteaεome may contribute, along with the induction of the transporters and MHC-clasε I proteins, to the enhancement of antigen preεentation. Methods Treatment of Cells with γ-IFN U937 cells (0.15 X 10 6 /ml) were grown in 150 cm 3 flasks, in RPMI 1640 medium containing 10% FCS and antibiotics at 37°C for 72 hours in the presence or absence of 1000 U/ml of human recombinant γ-IFN (kindly provided by Biogen, Inc. Cambridge, MA) . In preliminary studies, this concentration of γ-IFN was found to cauεe maximal changeε of proteasomal peptidaseε activitieε in U937 cells. Mutant (721.174) and wild-type (721) cellε were grown for three days in the presence or absence of 3,000 U/ml of γ-IFN, which was found to cauεe maximal changeε in peptidaεes activities. Peptidase Assays Hydrolysis of 125 I-human lactalbumin or ubiquitin- conjugated- 1 5 I-lactalbumin was assayed by measuring the production of radioactive peptideε εoluble in 10% trichloracetic acid (Wax an et al . , 1987). Proteaεome fractions or crude extracts (0.1 mg/ml) and purified proteasomes (5 μg/ml of 20S or 15 μg/ml of 20S) ; were incubated in the Buffer B (see Detailed Description of Figure 1) with radioactive substrates at 37°C for 60 or 120 minutes in the presence of 2 mM ATP or 5 U/ml of apyrase (to deεtroy residual ATP) . Nonradioactive lactalbumin (20 μg/ml) was added to reaction mixtures containing Ub- 125 I-lactalbumin. Preparation of Crude Cell Extracts and Proteosome- Enriched Fractions To prepare extracts, cellε were collected by centrifugation for 5 minuteε at 700 x g, waεhed twice, and reεuspended in Homogenization Buffer (50 mM Tris, 5 M MgCl 2 , 1 mM dithiothreitol (DTT), 2 mM ATP, 250 mM sucrose, pH 7.4). Cell suspensions were homogenized by several passages through a Dounce homogenizer (Wheaton) , followed by vortexing for 3 minutes with glass beads. After centrifugation at 10,000 x g for 20 minutes, the supernatant ("crude extract") was centrifuged for 1 hour at 100,000 x g to obtain a "microso e pellet". The "proteasome fraction" was obtained by spinning the post- microsomal supernatant for 5 hours at 100,000 x g. Pellets were resuspended in about 10 volumes of Buffer A (50 mM Tris, 5 mM MgCl 2 , 1 mM DTT, 2 mM ATP, 20% glycerol), homogenized and centrifuged at 14,000 rpm for 15 minutes. The supernatantε, which contained 20% of the protein in crude extractε, were adjuεted to a concentration of 1 mg/ml (Bradford, 1976) prior to assay of proteolytic activities, as were the crude extractε (after dialysis against Buffer A) . With each set of cultured cells, two sets of extracts were prepared and analyzed in parallel. The averages of results obtained with the two sets were taken as the result of a particular experiment. Detailed Description of Figure 1 The upper panel of Figure 1 εhows the hydrolysis of the indicated substrates (100 μM) by proteasome fractions from control cells and cellε treated with γ- IFN (Mamburg et al . , 1992) . Peptidase activities were determined by the release of 7-amino-4-methylcoumarin (MCA) or methoxynaphthylamine (MNA) from fluorogenic peptide substrateε (Iεchiura et al . , 1985) . Proteaεome fractions (final protein concentration: 50 μg/ml in 0.1 ml of Buffer B: 50 mM Triε, 5 mM MgCl 2 , 5 mM DTT, pH 7.4, containing 5 U/ml apyraεe) were incubated for 40 or 60 minutes at 37°C. The reaction was terminated by adding 1 ml of 1% sodium dodecyl sulphate. The increase in degradation of hydrophobic and baεic substrates was highly significant (P<0.01 with n=6 different experiments) . In the crude extracts, very similar changes (P<0.02, n=3) were observed with Suc-LLVY-MCA and Boc-LRR-MCA, while degradation of Cbz-LLE-MNA did not change significantly. Units = mM of substrate cleaved per mg of protein per hour. The lower panel of Figure 1 shows kinetic analysis (Lineweaver-Burk method) of rates of cleavage of the substrates (v) by the same proteasome fractions used above. Incubations were carried out as in upper panel, with varying subεtrate concentration (S) . V maχ for Suc- LLVY-MCA was 0.7 units and 1.6 with γ-IFN; for Boc-LRR- MCA, 0.3 units and 1.9 with γ-IFN; for Cbz-LLE-MNA, 0.3 units and 0.2 with γ-IFN. K-,, for Suc-LLVY-MCA was 0.2 mM and 0.3 M with γ-IFN; for Boc-LRR-MCA, 0.4 mM and 0.5 mM with γ-IFN; for Cbz-LLE-MNA, 0.10 mM and 0.06 mM with γ-IFN. Detailed Description of Figure 2 Figure 2 shows the effect of γ-IFN treatment on peptidase activities in 20S and 26S proteasomes purified from U937 cells. The results are representative of 3 different experiments. All differenceε between control preparationε and thoεe from γ-IFN-treated cells were statistically significant (P<0.01), but no significant difference was seen for Cbz-LLE-MNA cleavage by 2OS. Units and assay conditions are as in Figure 1, but the final concentrations of the proteaεomeε were 2.5-3.0 μg/ml (26S) or 8-10 μg/ml (20S) . 20S and 26S proteasomes were isolated from the proteaεome fraction by Q Sepharose anion exchange and Superose 6 gel filtration FPLC chromatographies (Pharmacia) (Driscoll and Goldberg, 1989; Matthews et al . , 1989). The 26S- rich fraction was identified by its ability to support the ATP-dependent degradation of Ub- 125 I-lactalbumin. Routinely, about 30 μg of 26S and 90 μg of 20S were obtained from 5 mg of proteasome fraction. Detailed Description of Figure 3 The upper panel of Figure 3 showε the difference in activities of the peptidases of proteasomes from wild- type (721) and MHC-deficient (721.174) lymphoblastoid cells. The rates of hydrolysis of three subεtrateε (100 μM) were meaεured in proteasome fractionε, aε done for Figure 1. Differences in all three activities were statistically significant (P<0.01, n=4 different preparations) . The lower panel of Figure 3 shows the kinetic analysis of cleavage for all the subεtrates at different concentrations (S) by the same proteasome fractions from wild-type and mutant cells. V maχ for Suc-LLVY-MCA was 6.5 unitε (wild-type) versus 2.4 (mutant) ; for Boc-LRR- MCA, 1.6 unitε vε 0.7; for Cbz-LLE-MNA, 0.7 units vε 1.2. K--, for Suc-LLVY-MCA waε 0.5 mM (wild-type) vs 0.3 mM (mutant) ; for Boc-LRR-MCA, 0.3 mM vs 0.5 mM; for Cbz- LLE-MNA, 0.4 mM (wild-type and mutant). Very similar results were obtained in two different experiments. Detailed Description of Figure 4 The left panel of Figure 4 shows the frequency of amino acidε at the carboxyl termini of 44 peptideε that bind MHC claεs I molecules. The data were collected from published sequenceε of peptides (Falk et al . , 1991; Guo et al . , 1992; Hunt et al . , 1992; Jardetzky et al . , 1991; Matεumura et al . , 1992). Theεe sequences were determined in different studies either by Edman degradation of individual peptideε that were eluted and purified from MHC claεε I moleculeε or by functional analysis of synthetic antigenic peptides and alignment to known MHC-binding motifs. The MHC class I molecules included in this analysis fall into two groups: K b , D b , K d , L d and HLA-A2.1, which bind peptides that generally have a hydrophobic C-terminal reεidue, and HLA-B27 and HLA-A 68, which bind peptideε that generally have a basic C-terminal residue. The right panel of Figure 4 shows the frequency of amino acids preceding the N-termini of MHC class I-bound peptideε. The protein sequences from which 39 of the peptides in the left panel were derived were available. Data were collected from the EMBL (Heidelberg, Germany) and National Biomedical Research Foundation (Bethesda, Md) databaseε. TABLE 1 Stimulation of the Trysin-like and Chvmotrysin-like Peptidases of Proteasomes by γ-IFN Activity (uM cleaved/mq/h Substrate Control + γ-IFN % increase Hydrophobic Suc-LLVY-MCA 230 820 +357 SUC-LY-MCA 90 210 +133 Suc-AAF-MCA 60 120 +100 Basic Boc-LRR-MCA 57 250 +439 Boc-GGR-MCA 3.4 5.2 +53 Cbz-GKR-MCA 2.8 6.0 +114 Degradation rates of subεtrateε (100 μM) by proteaεome fractionε of U937 cells were analysed as in Figure 1 (upper panel) . Presented are averages from 6 (Suc-LLVY- MCA and Boc-LRR-MCA) or 2 (all other substrateε) different experiments. TABLE 2 Comparison of v- -IFN- -Induced Changes in Peptidase Act: Lvities of Proteasomeε fro Wild-type and MHC -deficierit C< ≥llε Y-IFN- ■ induced Increase in Activity Substrates Wildl-type MHC-deficient Mutant units 1 (% change) units 1 (% change) Hvdrophobic Suc-LLVY-MCA 340 (+33) 120 (+18)* Suc-LY-MCA 66 (+34) * 10 (+26)* Suc-AAF-MCA 62 (+42)* 8 (+15)* Basic Boc-LRR-MCA 180 (+48)* nε Boc-GKR-MCA 1.6 (+28)* ns Cbz-GGR-MCA 1.2 (+34)* nε Acidic Cbz-LLE-MNA -40 (-24)* +80 (+35)* Wild-type cells were 721 and mutant cells were 721.174. With al hydrophobic and basic substrates, γ-IFN caused consistently (n=3) larger changes in the wild-type cells. Interferon consistently caused opposite effectε with the acidic subεtrate in the wild- type and mutant cellε. Data are differenceε between meanε for proteasome fractions. Activities were determined aε in Figure 1. 1 Unitε -= μM of εubstrate cleaved per mg of protein per hour. * P<0.02, * P<0.05, ns - not significant (T test) . Example 2 Inhibition of MHC-I Antigen Preεentation by a Defect in Ubiquitin Conjugation The rate-limiting step in the complete degradation of most cellular proteinε iε their covalent conjugation with ubiquitin (Herεhko and Ciechanover, 1992; Rechtsteiner, 1987) . This step marks the proteinε for rapid hydrolyεiε by a 26S (1500 kD) proteolytic complex that containε a 20S (700 kD) degradative particle called the proteasome (Goldberg, 1992; Goldberg and Rock, 1992) . In addition to completely hydrolyzing cell proteins, some syεtem in the cytoεol generateε antigenic peptideε from endogenouεly εyntheεized cellular and viral proteinε (Townsend et al . , 1985; Townsend et al . , 1986; Morrison et al . , 1986; Moore et al . , 1988; Spies and Demarε, 1991; Powis et al . , 1991) . These peptides bind to newly εyntheεized claεε I major hiεtocompatibility complex (MHC) moleculeε in the endoplasmic reticulum and peptide-claεε I complexes are then transported to the cell surface for presentation to cytotoxic T cells (Yewdell and Bennink, 1992; Monaco, 1992) . Precisely how antigenic peptides are produced iε unknown, although indirect evidence suggestε a role for proteolytic particles (LMP) closely resembling and perhaps identical to the 20S (700 kD) proteasome (Goldberg and Rock, 1992; Monaco 1992; Parham, 1990; Yang et al . , 1992) . Applicantε therefore teεted if the ATP-ubiquitin- dependent proteolytic εyεtem iε involved in MHC claεε I-restricted antigen (Ag) presentation. Using cellε that exhibit a temperature- sensitive defect in ubiquitin conjugation, Applicants have found that the nonpermisεive temperature inhibited clasε I-reεtricted presentation of ovalbumin (OVA) introduced into the cytosol, but did not affect presentation of an OVA peptide synthesized from a minigene. These results implicate the ubiquitin-dependent proteolytic pathway in the production of antigenic peptideε. Tε20 cellε, a chemically mutagenized variant of the Chineεe hamεter lung cell line E36, express a thermolabile ubiquitin-conjugating enzyme El (Kulka et al . , 1988). El catalyzes the ATP-dependent activation of ubiquitin, an essential first step in conjugation of ubiquitin to cellular proteins (Hershko and Ciechanover, 1992; Rechtsteiner, 1987). Ubiquitin-protein conjugation and protein degradation are reduced in ts20 cells at nonpermissive temperature (41°C) (Kulka et al . , 1988; Gropper et al . , 1991). El is irreversibly inactivated in ts20 cells at 41°C. To determine whether blocking ubiquitin conjugation affected class I-restricted Ag presentation, ts20 and E36 cells were transfected with the cDNA geneε for H-2K b and ICAM-1 to enable the cellε to preεent Ag to the OVA-εpecific, K b -reεtricted murine T-cell hybridoma line RF33.70 (Rock et al . , 1990) . The tranεfected cell lineε, ts20.10.2 (mutant) and E36.12.4 (wild-type), were incubated at 41°C or 37°C for 1 hour before introducing OVA into the cytosol by osmotic lysiε of pinoεomeε (Townεend et al . , 1986). Incubations during and after the introduction of OVA were performed at 37°C; this eliminated temperature aε a variable between the groupε during and after their expoεure to OVA. The cellε were allowed to procesε the Ag for 1 hour before Ag procesεing waε stopped by aldehyde fixation. Incubation at 41°C greatly decreased presentation of OVA by mutant (Figure 5A) but not wild-type (Figure 5B) cells. Several hypotheses can be εuggeεted to explain these results. These posεibilitieε were examined. The antigen preεenting cellε (APCε) above acquired OVA by pinocytoεiε (Townsend et al . , 1986) . Therefore, presentation of OVA by mutant cells could have been inhibited if exposure to 41°C blocked pinocytosis. Mutant cellε incubated at 41°C or 37°C did not differ in their uptake of extracellular fluid (0.94 ± 0.27 nl/10 6 cellε at 41°C verεuε 0.96 ± 0.47 nl/10 6 cellε at 37°C, mean ± SD of four experimentε) as determined by the uptake of poly(vinylpyrrolidone) (PVP) , a commonly used marker of fluid-phase pinocytosiε (Wiley and McKinley, 1987) . The incubation conditions with PVP were identical to those described above for OVA. Thus, the failure of mutant cellε to preεent OVA waε not due to a defect in pinocytoεis. Alternatively, the inability of mutant cellε incubated at 41°C to present cytoεolic OVA could have been due to a block in the generation or presentation of the antigenic OVA peptide-K b complexes. Incubation at 41°C did not affect the ability of mutant (Figure 5C) or wild-type (Figure 5D) cells to present exogenouε 0VA 257 - 264 peptide (amino acidε 257-264 of ovalbumin) added to the medium. Under theεe conditionε, the εynthetic 0VA 257 _ 264 peptide (which repreεentε the naturally proceεεed epitope from ovalbumin) binds directly to K b on the cell surface (Falk et al . , 1991; Rock et al . , 1992) . Therefore, presentation of OVA peptide-K b complexes at the APC surface was not significantly affected by blocking ubiquitin conjugation. After antigenic peptides bind to newly synthesized clasε I moleculeε, the peptide-claεε I complexes are transported through the Golgi complex to the cell surface for presentation to T cells. Therefore, Applicants examined the effects of 41°C on the εyntheεiε and tranεport of H-2K b in mutant cellε. Following 1 hour incubation at 41°C or 37°C, cellε were radiolabeled with [ 35 S]-methionine, and H-2K b molecules were sequentially immunoprecipitated with a mAb (Y-3) (Townsend et al . , 1990) that is specific for assembled K b moleculeε. This waε followed by precipitation with rabbit anti-heavy chain antisera (exon 8) (Smith and Barber, 1990) . Similar amounts of asεembled H-2K b were immunoprecipitated from mutant cellε incubated at the two temperatureε (Figure 6A) . Applicantε also found at both temperatures similar amounts of free heavy chains (Figure 6A) and B 2 -microglobulin that were potentially available for aεsembly with peptide. To analyze the transport of assembled K b molecules through the Golgi complex, cellε were incubated at 41°C or 37°C for 1 hour and then pulse-radiolabeled and chased at 37°C. H-2K b molecules were immunoprecipitated and subjected to two-dimensional IEF/SDS-PAGE. Temperature did not affect the degree of charge or molecular mass heterogeneity of K b molecules in either mutant or wild-type cells (Figure 6B) . Since changes in charge and molecular mass during the intracellular maturation of clasε I molecules are aεεociated with complex εugar additionε in the Golgi complex, theεe findingε indicate that assembly and transport of H-2K b occurred normally in the mutant cells following incubation at 41°C. Thiε suggested that inhibition of Ag presentation was due to a selective defect in the production of antigenic OVA peptides. To examine this point further, mutant cells, treated as described in Figure 5, were incubated with OVA or infected with a recombinant vaccinia virus (Ova 257 _ 264 Vac) containing a minigene encoding the 0VA 257 _ 26 peptide preceded by an initiating Met. Presentation of the endogenously-synthesized °VA 257 _ 264 peptide was unaffected by incubation at 41°C (Figure 7A) , while, as shown earlier, the presentation of OVA was inhibited (Figure 7B) . After one hour of infection, the presentation of 0VA 257 _ 264 + Kb by the vaccinia infected cells was considerably lesε than the presentation by cells that were incubated with OVA (compare the 37°C groups in Figure 7A and 7B) . Therefore, it is unlikely that overproduction of the 0VA 257 _ 264 peptide could account for the lack of effect of the 41°C incubation on presentation by the vaccinia- infected mutant cells (also see Detailed Description of Figure 7) . This finding indicates that the reduction in ubiquitin conjugation caused by incubating mutant cells at 41°C did not affect either the transport of 0VA 257 _ 264 peptide from the cytosol into the endoplasmic reticulum, the subsequent assembly of the 0VA 257 _ 264 peptide and H-2K b , or the transport of the class 1-peptide complex to the cell surface. The simplest interpretation of the present findings is that ubiquitin conjugation playε a critical role in the proceεsing of OVA for claεε I reεtricted preεentation. In related studies. Applicants found that OVA added to cell-free extracts was degraded by the ubiquitin-dependent pathway, since thiε process required ATP, ubiquitin, a proteasome fraction from rabbit reticulocytes, and free amino groupε on the ovalbumin. The presence of two MHC-encoded proteins in the proteasome has led to the hypothesis that the proteasome is involved in processing cellular and viral Ags (Parham, 1990; Yang et al . , 1992) . Recent data, however, indicate that theεe subunits are not esεential for claεε I-restricted Ag presentation (personal communication from Hammerling et al . ) . Since ubiquitin-conjugated proteins are degraded by the 26S proteasome complex (Goldberg, 1992; Goldberg and Rock, 1992) , these findingε directly implicate thiε structure in procesεing of an Ag for claεε I-reεtricted presentation. Thus, the immune system utilizes the cell's two primary and phylogenetically old degradative pathways to supply antigenic peptides: the ATP-ubiquitin-proteasome dependent pathway for clasε I and the vacuolar pathway for class II-restricted preεentation. Detailed Description of Figure 5 Figure 5 εhowε MHC claεε I-restricted presentation of OVA by mutant (ts20.10.2) and wild-type (E36.12.4) cellε. Ts20.10.2 (A and C) and E36.12.4 (B and D) cells were incubated at 41°C (filled) or 37°C (open) and then incubated with (A and B) or without (C and D) OVA. The indicated number of APCs incubated with OVA were cultured with RF33.70 (A and B) , or control APCε (5 x IO 4 ) were cultured with RF33.70 and the indicated concentration of 0VA 257 _ 264 peptide (C and D) . Ts20.10.2 (mutant) and E36. 12.4 (wild-type) cells (2 x IO 6 cells/ml) were incubated for 1 hour at 41°C or 37°C. For introducing OVA into the cytosol, the cells were incubated for 10 minutes at 37 °c with 20 mg/ml OVA in hypertonic media, treated with hypotonic media, and then washed with ice-cold, serum-free RPMI 1640, as previously described (Townsend et aJ . , 1986; Michalek et al . , 1991) . The washed APCs (2 x 10 6 cells/ml) were incubated for 1 hour at 37°C to allow for the expression of processed Ag on the cell surface. During this 37°C incubation, some recovery of ubiquitin conjugating activity (16 ± 10% mean ± SD of three experiments) did occur in the mutant cellε that had been previously incubated at 41°C. APCs were fixed with 1% paraformaldehyde to prevent further recovery of ubiquitin conjugation and/or Ag processing and added to duplicate microcultures (200 μl) with RF33.70 (10 5 cells), as previously described (Michalek et al . , 1991). Control APCs were as described above except OVA was omitted from the hypertonic media. RF33.70 is stimulated to produce IL-2 upon recognition of processed OVA + K b on the surface of APCε (Rock et al . , 1990b). The microcultures were prepared, incubated, and asεayed for IL-2 content as previously described (Michalek et al . , 1991). The H-2K b cDNA, pBG367-K b , was kindly provided by Dr. Gerald aneck. The vector pcDL-SRccr296 was provided by Dr. Naoko Arai (Takebe et al . , 1988). The H-2K b cDNA was subcloned into the Xhol/BamHI sites of the pcDL- SRα296 vector. Murine ICAM-I cDNA in the pH B APr-l-neo vector was provided by Drs. Hedrick and Brian (Siu et al . , 1989) . E36 and tε20 cells were cotranεfected with H-2K b and ICAM-I using the Lipofectin reagent (BRL) and εelection with G418 (GIBCO) were performed as previously described (Dang et al . , 1990) , except ts20 cells were grown at 37 °C following trans ection. G418-resiεtant cloneε were screened for expression of ICAM-I and H2K b with mAb YNl/1.7.4 (Dang et al . , 1990) and B8-24-3 (Kohler et al . , 1981), respectively. The transfected cell lines ts20.10.2 and E36.12.4 were pasεaged at 31°C and 37°C, respectively. Detailed Description of Figure 6 Figure 6 showε the effect of temperature on the synthesis and maturation of H-2K b . In Figure 6A, mutant (ts20.10.2) and wild-type (E36.12.4) cellε were incubated for 1 hour at 41°C or 37°C and then metabolically radiolabeled for 15 minutes at 37°C. H-2K b was sequentially immunoprecipitated with Y-3 (Jones and Janeway, 1981) followed by precipitation with anti-exon 8 (Smith and Barber, 1990) (a kind gift from Dr. Brian Barber, University of Toronto) and analyzed by one-dimensional SDS-PAGE. Y-3 recognizes a confor ational epitope formed by the αl and α2 domains of H-2K b and binds only assembled H-2K b molecules, whereas the anti-exon 8 antisera was raised against the C-terminal cytoplasmic domain of H-2K b (encoded by exon 8) and binds both assembled H2K molecules and free heavy chains. The positions of H-2K b heavy chains (H) and B 2 -microglobulin (L) and of the relative molecular asε standards (M r x IO- 3 ) are indicated in Figure 6. Forty million cells were incubated for 1 hour at 41°C or 37°C in methionine-free media and then radiolabeled for 15 minutes at 37°C with 0.7 mCi of [ 35 S]-methionine (>1000 Ci/mmol, NEN) . Detergent lysates, im unoprecipitations and SDS-PAGE were preformed essentially as described (Townsend et al . , 1990) , except that additional preclearing steps with IgGsorb (5 timeε) and PA-sepharoεe (1 time) were included before and after the first immunoprecipitation, respectively. In Figure 6B, ts20.10.2 and E36.12.4 cells were incubated for 1 hour at 41°C or 37°C, metabolically pulse-radiolabeled for 10 minutes at 37°C, and then chased for 50 minuteε at 37°C. H-2K b waε immunoprecipitated with Y-3 and analyzed by two-dimenεional IEF/SDS-PAGE. Immunoprecipitateε made at the 0 time point of the chaεe were analyzed and showed a single immature heavy chain spot, which chased into the more acidic and larger molecular maεε forms shown above. The heavy chains of H-2K b are indicated by the H, and the B 2 -microglobulin is indicated by the L. Cells were incubated and pulsed-radiolabeled for 10 minutes with [ 35 S]-methionine as described above. The cells were then chased in the presence of cold methionine (15 mg/ml) for 50 minutes at 37°C. Cell lysates were prepared and immunoprecipitations with Y-3 were performed as described above. For two-dimensional IEF/SDS-PAGE, the immunoprecipitates were analyzed by using the Immobiline DryStrip Kit (Pharmacia) . Gels were incubated in Autofluor (Nalional Diagnostics) , and radiolabeled proteins were visualized by autoradiography at -80°C. Detailed Description of Figure 7 Figue 7 εhows MHC class I-reεtricted presentation of endogenously-synthesized 0VA 25 _ 264 peptide. Ts20.10.2 cells were incubated at 41°C (filled) or 37°C (open) for 1 hour and then either infected with 0va 257 - 264 Vac for ■*- hour at 37 C C (Figure 7A) or incubated with OVA (Figure 7B) . The indicated number of APCs were incubated with RF33.70. Ts20.10.2 cellε (7 x IO 5 cells/ml) were incubated for 1 hour at 41°C or 37°C and then infected with recombinant vaccinia viruε (10 PFU/cell) for 1 hour at 37°C (Figure 7A) . Ts20.]0.2 cells were incubated at 41°C and 37°C, treated with OVA, and then incubated at 37°C as described above (see Detailed Description of Figure 5) . The APCs infected with vaccinia virus or incubated with OVA were fixed with 1% paraformaldehyde and incubated in microcultures with RF33.70 (IO 5 cellε) as described in the Detailed Description of Figure 5. The microcultures were prepared, incubated, and asεayed for IL-2 content aε also described therein. APCs infected with a recombinant vaccinia virus containing an influenza nucleoprotein gene did not stimulate RF33.70. Time-courεe experiments with Ova 257 _ 26 ac showed that the amount of 0VA 257 _ 26 peptide-K b complexes on the surface of cells infected for 1 hour was limiting because ceilε infected for 3 hours were 64-fold more efficient at stimulating RF33.70. Ova 25 _ 264 Vac was constructed by inserting a synthetic oligonucleotide behind the vaccinia virus p7.5 early/late promoter in pScll (Chakrabati et al . , 1985), which was modified such that the restriction sites Sail and iVotl were substituted for the Smal site. The oligonucleotide: 5'-TCGACCACCATGTCTATAATAAACTTTGAGAAGTTATAGTGACCATGGGC-3 ' 3 '-GGTGGTACAGATATTATTTGAAACTCTTCAATATCACTGGTACCCGCCGG-5' (Seq. ID #1), conεisted of a Sail site, Kozak's consensus sequence for efficient translation, an initiation codon, nucleotides encoding the peptide S I I N F E K L (Seq. ID #2) , two stop codons, and a Λ ' otI εite, which is not present in pSCll and therefore provides a simple method for determining the presence of the insert in the plasmid. After strand annealing, the synthetic oligonucleotide was phosphorylated with T4 polynucleotide kinase. pSCll waε digested with Sa l and Notl, purified, and ligated with the oligonucleotide using T4 ligase. Homologous recombination of the plasmid with vaccinia virus, selection, and propagation of recombinants was then performed aε described (Moεε and Earl, 1991) . Example 3 Inhibition of MHC-1 Preεentation By Chvmoεtatin ANTIGEN PRESENTING CELLS The antigen-presenting cellε (APCε) in this experiment were a mouse B lymphoblastoid cell line (LB27.4) that was passaged in media supplemented with normal mouse serum (1%) and lipopolysaccharide (10 μg/ml X 72 hours) . Except for the inclusion of the lipopolysaccharide, this is described in Rock et al . , 1990b. INHIBITOR TREATMENT AND ANTIGEN LOADING The LB27.4 cells were washed serum free and incubated in the presence or absence of chymostatin (200 μg/ml; Boεhringer Mannheim, Indianapoliε, IN) for one hour at 37°C. The cells were then resuεpended in Electroporation buffer (phoεphate buffered εaline, 1 mM Hepeε, 0.4 M mannitol; chymoεtatin was also added to the appropriate groupε) containing either the antigen ovalbumin (30 mg/ml) or a εynthetic peptide corresponding to amino acids 257-264 of ovalbumin. The resuspended cells were then subjected to electroporation on the CELL-PORATOR from Gibco BRL (Gaitherεburg, MD) at 4°C with εetting of high ohms, capacitance 1180. The cells were washed 4 times at 4°C and incubated at 37°C for two hourε in the continued presence or absence of chymostatin. After this two hour incubation, the cellε were fixed with 1% paraformaldehyde for 10 minutes at room temperature, followed by washing. ANTIGEN PRESENTATION ASSAY The indicated number of fixed antigen-presenting cells were incubated with 10 5 RF33.70 cells (an OVA + K b εpecific T-T hybridoma) in duplicate 200 μl microcultures. After 18 hours at 37°C, an aliquot (100 μl) of culture supernatant was harvested and aεεayed for IL-2 content using HT-2 cells, as described in Rock et al . , 1990a and 1990b. Detailed Description of Figure 8 Figure 8 shows MHC-I presentation of ovalbumin (OVA left panel) and an ovalbumin peptide (right panel) . In the left panel, APCs were treated with (open circles) or without (closed circles) chymoεtatin and electroporated with ovalbumin. In the right panel. APCs were treated with (open circles) or without (closed circles) chymostatin and electroporated with ovalbumin peptide. The left panel demonstrates that chymostatin inhibits the presentation of ovalbumin with class I MHC molecules. The right panel demonstrates that chymostatin does not inhibit the presentation of electroporated peptide. This result indicateε that the inhibition of antigen preεentation is occurring through chymostatin inhibition of the processing of the ovalbumin protein into the ovalbumin peptide. EXAMPLE 4 Isolation of an Endogenous Inhibitor of the Proteasome As described in Example 7, a 40 kDa polypeptide regulator of the proteasome, which inhibits the proteasome's proteolytic activities, has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis. The isolated inhibitor exists as a 250 kDa ultimer and is quite labile (at 42°C) . It can be stabilized by the addition of ATP or a non-hydrolyzable ATP analog, although the purified inhibitor does not require ATP to inhibit proteasome function and lacks ATPase activity. The inhibitor has been εhown to correεpond to an eεεential component of the 1500 kDa proteolytic complex. If reticulocyteε are depleted of ATP, the 1500 kDa UCDEN is not found. Instead, Ganoth et al . identified three components, designated CF-1, CF-2 and CF-3, (J. Biol . Chem . 263:12412-12419 (1988)). The inhibitor isolated as described herein appears identical to CF-2 by many criteria. These findings indicate the idea that the inhibitor plays a role in the ATP-dependent mechanism of the UCDEN complex. It is possible, for example, that during protein breakdown, within the 1500 kDa complex, ATP hydrolysis leads to functional release of the 40 kDa inhibitor, temporarily allowing proteasome activity, and that ubiquitinated proteins trigger this mechanism. The purified factor has been shown to inhibit hydrolysis by the proteasome of both a fluorogenic tetrapeptide and protein εubεtrateε. When the inhibitor, the proteasome and partially purified CF-l were mixed in the presence of ATP and Mg + , the 1500 kDa complex was reconstituted and degradation of Ub- 125 I- lysozyme occurred. Isolation of this inhibitor of the multiple peptidaεe activities of the proteasome makes available an attractive site for pharmacological intervention. As deεcribed subsequently, this provides a natural inhibitor whose structural and functional featureε can be assessed to provide information useful in developing proteasome inhibitors. Materials and Methodε DEAE-cellulose (DE-52) , CM-cellulose (CM-52), and phosphocelluloεe (Pll) were obtained from Whatman. Ub- conjugating enzymeε (El, E2 and E3) were iεolated using Ub-sepharose affinity column chromatography (Hershko et al . , J . Biol . Chem . 258:8206-8215 (1983)), and were used to prepare Ub- 125 I-lysozyme conjugates (Hershko and Heller, Biochem . Biophys . Res . Comm . 128:1079-1086 (1985)) . All other materials used were as deεcribed in the previouε exa pleε. Purification Rabbit reticulocyteε induced by phenylhydrazine injection were prepared (as described previously or purchaεed from Green Hectareε (Oregon, WI) . They were depleted of ATP by incubation with 2, 4-dir." trop ' .-enol and 2-deoxyglucose as described (Ciechanover et al . , Biochem . Biophys . Res . Comm . 81:1100-1105 (1978)) . Lyεates were then prepared and subjected to DE-52 chromatography. The protein eluted with 0.5M KC1 (Hershko et al . , J . Biol . Che . , 258:8206-8214 (1983)) was concentrated using ammonium sulfate to 80% saturation, centrifugated at 10,000 x g for 20 minutes, and suspended in 20 mM Tris-HCl (pH 7.6) , 1 mM DTT (Buffer A) . Following extensive dialysis against the same buffer, the protein (fraction II) was either stored at -80°C in 0.5 mM ATP or fractionated further. Fraction II (-200 mg) was applied to a Ub-sepharose column, and the Ub-conjugating enzymes were specifically eluted (Hershko et al . , J . Biol . Chem . , 258:8206-8214 (1983)) and used in making Ub-lysozyme (Hershko and Heller, Bi ochen . Biophys . Res . Comm . 128:1079-1086 (1985)) . The unabsorbed fraction was brought to 38% saturation using ammonium sulfate and mixed for 20 minutes, as described by Ganoth et al . (Ganoth et al . , J. Biol . Chem . 263:12412-12419 (1988)). The precipitated proteins were collected by centrifugation at 10,000 x g for 15 minutes. The pellet was resuspended in Buffer A and brought again to 38% saturation with ammonium sulfate. The precipitated material was collected as above and then suspended in Buffer A containing 10% glycerol. After dialysis against this buffer, the 0-38% pellet was chromatographed on a Mono-Q anion exchange column equilibrated with Buffer A containing 10% glycerol. The protein was eluted using a 60 ml linear NaCl gradient from 20 to 400 mM. Fractions which inhibited the peptidase activity of the proteasome were pooled, concentrated, and then chromatographed on a Superose 6 (HR 10/30) gel filtration column equilibrated in Buffer A containing 100 mM NaCl and 0.2 mM ATP. The column was run at a flow rate of 0.2 ml/minute, and 1 ml fractionε were collected. Further purification of the inhibitor waε achieved by a second more narrow Mono-Q chromatographic gradient (from 50 to 300 mM NaCl) , which yielded a sharp peak of inhibitor where only the 40 kDa band was viεible after SDS-PAGE and Coomaεie εtaining. Fractions with inhibitory activity against the proteaεome were pooled and dialyzed against Buffer B which contained 20 mM KH 2 P0 4 (pH 6.5) , 10% glycerol, 1 mM DTT and 1 mM ATP. The sample was then applied to a 2 ml phoεphocellulose column equilibrated in Buffer B. The column was washed with 4 ml of this buffer, followed by 4 ml of this buffer, followed by 4 ml of Buffer B containing either 20, 50, 100, 400 or 600 mM NaCl. To obtain partially pure CF-1, the Mono-Q fractions that eluted from 100 to 240 mM NaCl were pooled, concentrated to 1 ml and applied to a superoεe 6 column equilibrated in Buffer A containing 100 mM NaCl and 0.2 mM ATP. The fractions eluting at approximately 600 kDa were used as the CF-1 containing fraction. The proteasome was isolated from the supernatants of the two 38% ammonium sulfate precipitations. The supernatants were brought to 80% saturation with ammonium sulfate and mixed for 20 minutes. The precipitated protein was collected by centrifugation, resuspended in Buffer A, and dialyzed extensively against this buffer. The proteasome was isolated by Mono-Q anion exchange chromatography followed by gel filtration on superoεe 6 as described previously (Driεcoll and Goldberg, Proc . Natl . Acad . Sci . , USA 86:789-791 (1989)). Tiie 1,500 kDa proteolytic complex waε generated by incubating reticulocyte fraction II at 37°C for 30 minuteε in the preεence of 2 mM ATP, 5 mM MgCl 2 in 50 mM Triε-HCl (pH 7.6) . After precipitation with ammonium sulfate to 38% saturation, the pellet was collected at 10,000 x g for 10 minutes, suspended in Buffer A, and isolated by Mono-Q anion exchange and εuperose 6 chromatography. Assays Inhibition of the proteaεome was measured by preincubating individual column fractions with the proteasome in the presence of 1 mM ATP at 37°C for 10 minutes. After preincubation, the reaction tubes were placed on ice, and either 125 I-lysozyme or Suc-LLVY-MCA was added. Reactions were carried out at 37°C for 60 minutes with 125 I-lysozyme or 10 minutes with Suc-LLVY- MCA. Protein hydrolysiε waε assayed by measuring production of radioactivity soluble in 10% trichloroacetic acid, and peptide hydrolysis by the release of methylcoumaryl-7-amide (Driscoll and Goldberg, Proc . Natl . Acad . Sci . , USA 86:789-791 (1989)). Degradation of Ub-conjugated 125 -I-lysozyme was asεayed at 27°C for 60 inuteε. Reactionε contained either 5 M EDTA or 2 mM ATP and 5 mM MgCl 2 and were terminated by adding 10% trichloroacetic acid. RESULTS Iεolation of the Inhibitor To understand how the proteasome is regulated in vivo and how it functions in the Ub-conjugate-degrading complex, Applicants attempted to isolate factors which influence its activity. Reticulocyte fraction II was separated using ammonium sulfate into fractions which precipitated with either 0-38% or 40-80%. The latter fraction was used to iεolated proteasomes. The particles (obtained in this way from ATP-depleted reticulocytes) showed appreciable activity against 125 I- lyεozyme and Suc-LLVY-MCA which waε independent of ATP (Eytan et al . , Proc . Natl . Acad . Sci . , USA 86:7751-7755 (1989); Driscoll and Goldberg, J . Biol . Chem . 265:4789- 4792 (1990)) . Neither the proteasome nor the 0-38% fraction showed significant activity against Ub- conjugated 125 I-lysozyme (Eytan et al . , Proc . Natl . Acad . Sci . , USA 86:7751-7755 (1989) ; Driscoll and Goldberg, J . Biol . Chem .265: 789-4792 (1990)). However, as reported previously, ATP-dependent degradation of the ubiquitinated lysozyme was obεerved after the protease and the 0-38%fraction were preincubated together in the presence of ATP. The 0-38% precipitated material was then separated using Mono-Q anion exchange and each fraction asεayed for its ability to influence the proteasome activity against Suc-LLVY-MCA or 125 I-lysozyme. Column fractions were preincubated with the proteasome for 10 minutes and then either substrate was added. None of the column fractions by itεelf showed significant hydrolytic activity. A peak of inhibitory activity was eluted around 240 to 280 mM NaCl. It significantly decreased proteolytic activity against both substrateε. Hydrolysis of lysozyme and the peptide was inhibited to a similar extent. To purify the inhibitory activity further, the active fractionε were pooled and chromatographed by gel filtration. The inhibitor eluted as a sharp peak with an apparent molecular weight of about 100-150 kDa. The active fractions were then pooled and aεεayed for their ability to inhibit the εubεtrate hydrolyzing activities of the proteaεomes. With increasing inhibitor concentration, proteasome activity decreased in a linear manner with both 125 I-lysozyme and Suc-LLVY- MCA as substrates, although the degree of the inhibition was highly variable between preparations. The Inhibitor Iε A Component of the l,500kDa Proteolytic Complex Like the inhibitor, one component of the 1,500 kDa proteolytic complex (CF-2) has been reported to have a molecular weight of about 250 kDa. To test if the inhibitor corresponds to CF-2, the inhibitor obtained by gel filtration was subjected to phosphocellulose chromatography. Eytan et al . had noted that CF-2 has little affinity for phosphocellulose and elutes with leεs than 100 mM NaCl. Accordingly, Applicants found that the inhibitory activity waε recovered in the flow through and 20 mM NaCl eluate (i.e., in the region where CF-2 activity was reported) . Individual phosphocellulose fractions were then assayed for their ability to reconstitute degradation of ubiquitinated lyεozyme. Individually or combined, the proteaεome and CF-1 containing fraction did not support rapid breakdown of ubiquitinated lysozyme. However, when this mixture was combined with the peak of the inhibitor activity, the rate of Ub- 125 I-lysozyme degradation increased sharply. No other phosphocellulose fractions stimulated this procesε. These results suggeεt εtrongly that the inhibitor correεpondε to CF-2 and thus is eεεential for hydrolyεis of Ub-ligated proteinε. One unuεual property of CF-2 is that it iε quite labile upon heating to 42°C, but iε εtabilized by ATP (Ganoth et al . , J. Biol . Chem . 263:12412-12419 (1988)) . To test further if the inhibitor of the proteaεome corresponds to CF-2, the purified inhibitor was preincubated at 42 *C with or without ATP or the nonhydrolyzable analog, AMPPNP. The proteaεome was added and after 10 minutes, peptidase activity waε assayed. The degree of inhibition decreased rapidly during preincubation without nucleotide added. The preεence of either ATP or AMPPNP prevented this loss of activity. Furthermore, the ability of this material to reconstitute degradation of Ub-conjugated lysozyme also decreased rapidly during incubation of 42°C, and the addition of ATP or AMPPNP (not shown) prevented this activation. Since the inhibition and reconεtitution of Ub-conjugate degradation showed similar inactivation kinetics and were stabilized similarly by ATP, theεe two functionε probably reεide in a single molecule which appears to bind ATP. Although ATP stabilizes the inhibitory factor, it is not essential for inhibition of the proteasome. After preincubation of the inhibitor with proteasome for up to 20 minutes with or without ATP, a similar degree of inhibition was observed. Nevertheless, because of the stabilization by ATP, this nucleotide was routinely added to all incubations. When analyzed by SDS-PAGE, the inhibitor preparations showed a major band of 40 kDa. To test whether this 40 kDa subunit corresponded to any subunit of the 1,500 kDa complex, the 1,500 kDa complex was formed by incubation of fraction II with Mg -ATP and isolated by anion exchange and gel filtration chromatography. SDS-PAGE of these active fractionε indicated many polypeptideε εimilar to those previously reported for thiε complex (Hough et al . , J. Biol . Chem . 262:8303-8313 (1987) ; Ganoth et al . , J . Biol . Chem . 263:12412-125419 (1988); Eytan, E. et al . , Prcc . Natl . Acad . Sci . USA 86:7751-7755 (1989)) . However, a readily apparent band of 40 kDa was evident in this fraction. 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SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: THE PRESIDENT AND FELLOWS OF HARVARD COLLEGE AND DANA FARBER CANCER INSTITUTE (ii) TITLE OF INVENTION: Role of ATP-Ubiquitin-Dependent Proteolysis in MHC-1 Restricted Antigen Presentation And Inhibitors Thereof (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox (B) STREET: 1100 New York Avenue, Suite 600 (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: USA (F) ZIP: 20005-3934 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: TO BE ASSIGNED (B) FILING DATE: 27-JAN-1994 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Goldstein, Jorge A. (B) REGISTRATION NUMBER: 29,021 (C) REFERENCE/DOCKET NUMBER: 1448.003PC00 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (202) 371-2600 (B) TELEFAX: (202) 371-2540 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : TCGACCACCA TGTCTATAAT AAACTTTGAG AAGTTATAGT GACCATGGGC 50 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Ser lie lie Asn Phe Glu Lys Leu 1 5 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (ix) FEATURE: (A) NAME/KEY: Modified-εite (B) LOCATION: 1..4 (D) OTHER INFORMATION: /note= "The sequence Leu Val Val Tyr is part of a modified peptide having the structure Sue-Leu Val Val Tyr-MCA." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : Leu Leu Val Tyr 1

Claims: CLAIMS The invention claimed iε: 1. A method for inhibiting MHC-I antigen presentation in an antigen-presenting cell, comprising contacting the cell with an inhibitor that inhibitε proteolysis of intracellular proteins by the ATP- ubiquitin-dependent pathway, under conditions suitable for the inhibitor to enter the cell. 2. The method of Claim 1, wherein the inhibitor inhibits a peptidase of the proteasomes, said peptidase having an activity which iε selected from the group consisting of: a) cleavage after basic residues; b) cleavage after hydrophobic residueε; and c) a combination' of a) and b) . 3. The method of Claim 2, wherein the inhibitor is a peptide aldehyde. 4. The method of Claim 3, wherein the peptide aldehyde is εelected from the group conεiεting of chymostatin and leupeptin. 5. The method of Claim 1, wherein the inhibitor inhibits ubiquitin conjugation of the intracellular proteins. 6. The method of Claim 5, wherein the inhibitor inhibits a protein εelected from the group conεi ' εting of: a) El, alεo referred to aε ubiquitin activating enzyme; b) E2 , also referred to aε ubiquitin-carrier protein; and c) E3, alεo referred to as ubiquitin-protein ligase. 7. The method of Claim 6, wherein the inhibitor of El is a substrate analog of ubiquitin adenylate. 8. A method for inhibiting cytolytic immune responses in a mammalian tissue, comprising contacting the tissue with an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the tissue. 9. The method of Claim 8, wherein the inhibitor inhibits a step in the proteolysis selected from the group consisting of: a) cleavage by a peptidase of the proteasomes, which is selected from the group consisting of cleavage after basic residues and cleavage after hydrophobic residueε; b) ubiquitin conjugation; and c) a combination of a) and b) . 10. The method of Claim 9, wherein the inhibitor inhibitε a protein εelected from the group consisting of: a) El, also referred to as ubiquitin activating enzyme; b) E2 , also referred to aε ubiquitin-carrier protein; and c) E3, alεo referred to aε ubiquitin-protein ligase. 11. A method for inhibiting cytolytic immune responεes in an individual, comprising administering to the individual an inhibitor that inhibits proteolyεis of intracellular proteins by the ATP-ubiquitin dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the individual. 12. The method of Claim 11, wherein the inhibitor inhibitε a step in the proteolysis selected from the group consisting of: a) cleavage by a peptidase of the proteaεomes, which is εelected from the group consisting of cleavage after basic residues and cleavage after hydrophobic residues; b) ubiquitin conjugation; and c) a combination of a) and b) . 13. A method of therapy or prevention of an autoimmune disease in an individual, comprising administering to the individual an inhibitor that inhibits proteolysis of intracellular proteins by the ATP- ubiquitin-dependent pathway, under conditions suitable for the inhibitor to enter antigen- presenting cells in the individual. 14. The method of Claim 13, wherein the inhibitor inhibits a step in the proteolysis selected from the group consisting of: a) cleavage by a peptidase of the proteasomeε, which is εelected from the group conεiεting of cleavage after basic residues and cleavage after hydrophobic residues; b) ubiquitin conjugation; and c) a combination of a) and b) . 15. A method for reducing rejection of foreign tissue by an individual, comprising administering to the individual an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin- dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the individual. 16. The method of Claim 15, wherein the inhibitor inhibits a εtep in the proteolysiε εelected from the group consiεting of: a) cleavage by a peptidaεe of the proteasomes, which is εelected from the group consisting of cleavage after baεic reεidueε and cleavage after hydrophobic reεidueε; b) ubiquitin conjugation; and c) a combination of a) and b) . 17. The method of Claim 15, wherein the foreign tissue is a transplanted organ or graft.

Inventors: GOLDBERG ARTHUR L, ROCK KENNETH L, GOLDBERG, ARTHUR, L., ROCK, KENNETH, L.
Applicants: DANA FARBER CANCER INST INC, HARVARD COLLEGE, DANA FARBER CANCER INSTITUTE, THE PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Application year: 1994
Main classification: A61K37
IPC codes: A61K38, A61K45, A61P37, A61P43, C07K14